Research article Received: 4 September 2013,
Revised: 3 November 2013,
Accepted: 18 January 2014
Published online in Wiley Online Library: 10 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2650
A new and highly sensitive resonance Rayleigh scattering assay for lysozyme using aptamer– nanogold as a probe Lu Ma, Xinghui Zhang, Aihui Liang, Qingye Liu and Zhiliang Jiang* ABSTRACT: Gold nanoparticles (GN), 10 nm in size, were modified by using lysozyme aptamer (Apt) to obtain a stable Apt–GN probe in pH 8.05 Tris/HCl buffer solutions containing 0.04 mol/L NaCl. Upon addition of lysozyme (LYS), it reacted with the Apt of the probe to form a very stable Apt–LYS complex and to release GNs, which aggregated to form large clusters with a resonance Rayleigh scattering (RRS) peak at 368 nm. The enhanced peak intensity, ΔI, was linear to the LYS concentration in the range 0.2–5.2 nmol/L, with a detection limit of 0.05 nmol/L. The influence of foreign substance was tested, and the results showed that this RRS method has high selectivity. This Apt–GN RRS method was applied to the analysis of LYS in a real sample, with satisfactory results. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: lysozyme; aptamer; gold nanoparticle; resonance Rayleigh scattering
Introduction
Luminescence 2014; 29: 1003–1007
Experimental Apparatus and reagents A F-7000 Hitachi fluorescence spectrometer (Hitachi Co., Tokyo, Japan), a TU-1901 double-beam UV/vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China), a JSM-6380 LV scanning electron microscope (Japan), a JEM-2100 F field emission transmission electron microscope (Japan), a 79-1 magnetic heating stirrer (Zhongda Instrumental Plant, Jiangsu, China), and a SK8200LH ultrasonic reactor (Kedao Ultrasonic Instruments Ltd, Shanghai, China) were used. A 3.49 μmol/L stock solution of LYS was prepared by dissolving 0.0500 g of LYS in 10 mL of water, and a 0.174 μmol/L LYS working solution was used. LYS aptamer (Sangon Biotech Co., Ltd.) with a sequence of 5′-CAGTGTATCGAATTCATCAGGGCTA AAGAGTGCAGAGTTACT-3′ was used at a concentration of 100 μmol/L; 2.0 mol/L NaCl, 1.0% HAuCl4 and 1.0% trisodium citrate were also used. The gold nanoparticle (GN) was synthesized by reduction of HAuCl4 using trisodium citrate. Doubly distilled water (50 mL) was added to a flask and brought to the boil under stirring. * Correspondence to: Z. Jiang, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Conservation of Education Ministry and Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, Guangxi Normal University, Guilin 541004, China. Tel: +86-07735846141; Fax: +86-07735846201. E-mail: [email protected] Key Laboratory of Ecology of Rare and Endangered Species and Environmental Conservation of Education Ministry and Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, Guangxi Normal University, Guilin541004, China
Copyright © 2014 John Wiley & Sons, Ltd.
1003
Lysozyme (LYS), also known as muramidase or N-acetylmuramide glycanohydrolase, is an alkaline globulin that consists of 129 amino acid residues, and is a powerful bactericide found in nasal mucus by Fleming in 1922 (1). It exists in mammalian body fluids including tears, saliva, plasma, urine, milk, microorganisms and egg white at a high level. LYS has antimicrobial and bactericidal effects; it selectively dissolves the outer membrane of bacteria, destroying their bioactivity. Because it is safe and non-toxic, LYS has been used as a biological preservative in the food industry (2). In addition, LYS also has anti-inflammatory, anti-virus, immune-boosting and other pharmacological activities, and it has also been applied as a potential marker in the diagnosis of disease (3,4). Therefore, it is important to develop a simple, rapid and sensitive method for the determination of LYS. To date, several methods have been used to determine LYS, including spectrophotometry (5), electrochemistry (6,7), surface plasmon resonance (SPR) (8,9), chemiluminescence (10), fluorescence (11) and resonance Rayleigh scattering (RRS) (12,13). However, these methods have some limitations due to their complicated modes of operation, poor sensitivity or poor selectivity. As aptamer (Apt) is selected from a synthetic and random sequence library in vitro via a combinatorial chemistry technique known as SELEX. Advantages of SELEX include ease of preparation, high stability, ease of modification, and a wide range of binding target molecules such as metal ions, small molecules, proteins and bacteria (14–17). It has been an important development in analytical chemistry. RRS spectroscopy is sensitive, rapid and simple, and has been used in a wide range of applications in different fields such as biochemistry, analytical chemistry and nanomaterial research (18–24). Combined with Apt-modified nanogold (GN), RRS has been used in the detection of metal ions (25), melamine, ATP (26,27) and thrombin (28). As far as we know, there is no report of the use of RRS for the detection of LYS, based on an Apt-modified GN probe. In this paper, a novel and simple
Apt–GN RRS assay was proposed to detect LYS, based on the Apt–GN reaction and the RRS effect of GN aggregates.
L. Ma et al. 3500 3000
Intensity (a.u.)
Then, 3.5 mL of 1% trisodium citrate and 0.5 mL of 1% HAuCl4 were added rapidly to the boiling water. After boiling for 10 min with stirring, the color changed form lilac to purple to red. The mixture was cooled to room temperature with stirring, and then diluted to 50 mL. The GN concentration was 58.0 μg/mL. Apt–GN was prepared as follows: 2.0 mL of a 58.0 μg/mL GN solution was added to 0.8 mL of a 1 μmol/L Apt solution, mixed well and set aside for 10 min. The Apt–GN concentration, calculated as Apt, was 0.28 μmol/L. All reagents were of analytical grade and the water was doubly distilled.
g
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a
1500 1000 500 0 200
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300
400
500
600
Wavelength (nm)
Results and discussion Principle Apt combined with GN to form the Apt–GN probe via electrostatic forces, van der Waal’s forces and intermolecular forces, and the probe was stable in high-concentration salts. Upon addition, LYS reacted with the probe to form a very stable Apt–LYS complex and to release GNs. Under the action of NaCl, the GNs aggregated to form large clusters that caused the enhancement of RRS intensity. The number of released GNs increased as the concentration of LYS increased, the GN aggregates also became larger, and the RRS intensity increased accordingly. Based on this, a RRS assay could be developed for the detection of trace LYS (Fig. 1). RRS spectra In pH 8.05 Tris/HCl buffer solution and in the presence of NaCl, the RRS signal of the Apt–GN probe was weak (Fig. 2). Upon addition of LYS, the probe combined with LYS specifically, and the released NGs aggregated to form a large cluster, the color changed from red to purple, and the RRS intensity increased at 285, 368 and 525 nm (Fig. 2). Because the light sources in the apparatus have strong emission at wavelengths of 285 and 368 nm, the system has two RRS peaks caused by the light source at both wavelengths, and the peak at 525 nm was due
Figure 2. RRS spectra of LYS–Apt–GN systems. (a) 39.2 nmol/L Apt–GN + Tris/HCl (pH 8.05) + 40 mmol/L NaCl; (b) a + 0.4 nmol/L LYS; (c) a + 1.7 nmol/L LYS; (d) a + 2.6 nmol/L LYS; (e) a + 3.5 nmol/L LYS; (f) a + 4.4 nmol/L LYS; (g) a + 5.2 nmol/L LYS.
to the GNs. The peak at 525 nm is not sensitive, whereas the peak at 368 nm is most sensitive and has good linearity. Thus, a wavelength of 368 nm was chosen. Absorption spectra There is only an SPR absorption peak at 519 nm for GN particles. After modification by Apt, the SPR peak does not change (Fig. 3a), and the absorption is very low due to the low concentration of NGs (5.8 μg/mL Au). Upon addition, LYS reacted with the Apt in the probe to form a stable complex, which exhibited a new absorption peak at 620 nm. As the concentration of LYS was increased, the absorption increased at about 620 nm (Fig. 3). The red-shift and broadening of the SPR peak showed that NGs were aggregated into large clusters in the solution system. Electron microscope A 1.5 mL aptamer reaction solution was obtained, and centrifuged for 20 min at 15,000 rpm. After the supernatant had been removed, the precipitate was diluted with 1.5 mL water and dispersed by ultrasound for 30 min; the process was repeated twice. A 2.0 μL aliquot of solution was added to a silicon chip and observed by scanning electron microscopy. It 0.14 0.12 0.1
Abs
Two hundred and eighty microliters of 0.28 μmol/L Apt–GN, 50 μL of Tris/HCl (pH 8.05) and a known volume of LYS solution were added to a 5 mL marked test tube and the mixture was diluted to 1.0 mL, then 40 μL of 2.0 mol/L NaCl was added and the mixture was diluted to 2.0 mL. RRS spectra were recorded by synchronous scanning of the excited wavelength λex and emission wavelength λem (λex – λem = Δλ = 0), a photomultiplier tube (PMT) voltage of 450 V was used, and excited and emission slit width of 5 nm were used on the fluorescence spectrophotometer. The RRS intensity at 368 nm (I368) and the blank value (I368)0 without LYS were recorded. The value of ΔI = I368 – (I368)0 was calculated.
0.08 0.06 0.04 0.02 0 300
400
500
600
700
800
wavelength (nm)
1004
Figure 1. Principle of the Apt–RRS method for LYS.
wileyonlinelibrary.com/journal/luminescence
Figure 3. Absorption spectra of LYS–Apt–GN systems. (a) 39.2 nmol/L Apt–GN + Tris/HCl (pH 8.05) + 40 mmol/L NaCl; (b) a + 0.4 nmol/L LYS; (c) a + 1.7 nmol/L LYS; (d) a + 3.5 nmol/L LYS; (e) a + 4.4 nmol/L LYS; (f) a + 5.2 nmol/L LYS.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1003–1007
Apt-AuNP RRS method for LYS 3500 3000 2500 2000
I
was shown that the Apt–GN probe was dispersed in solution (Fig. 4a), LYS reacted with the Apt in the probe to form a stable complex and to release GNs, which aggregated to form large clusters under the action of NaCl (Fig. 4b). Transmission electron microscopy (Fig. 5) also indicated that the Apt–GN probe is spherical, with an average size of 10 nm and is dispersed in solution.
1500
Preparation of Apt–GN and its concentration selection A 200 μL aliquot of a 58.0 μg/mL GN solution was used to test the impact of Apt on the system I368. The Apt was not enough to protect GN when the Apt concentration was < 40 nmol/L, and GNs were aggregated into a large cluster that caused the increase in RRS intensity (Fig. 6). When the Apt concentration was 40 nmol/L, it was just enough to modify GN, and the Apt–GN complex was stable in solution. Thus, 40 nmol/L was the minimum amount of Apt that stabilized 200 μL GN. The effect of the Apt–GN concentration was also considered, and 280 μL of 0.28 μmol/L Apt–GN was chosen for use.
1000 500 0
10
20
30
40
50
c (nmol/L) Figure 6. Effect of the Apt concentration on ΔI368nm 5.8 μg/mL GN + 2.5 mmol/L Tris/HCl (pH 8.05) + 40 mmol/L NaCl.
Effect of pH Buffer solutions including acetate, phosphate and Tris/HCl were examined. Tris/HCl buffer solution, giving biggest ΔI value, was selected for use. The effect of pH on the system was tested (Fig. 7). When the pH was 8.05, the ΔI of the system was maximal, so pH 8.05 was chosen for use. The effect of Tris/HCl concentration was also tested, when the Tris/HCl concentration was 1.25 mmol/L, ΔI reached its maximum value. Therefore, a 1.25 mmol/L pH 8.05 Tris/HCl buffer was chosen.
a
Figure 7. Effect of pH on ΔI368nm 39.2 nmol/L Apt–GN + 3.5 nmol/L LYS + 40 mmol/L NaCl.
b
Effect of NaCl concentration The effect of NaCl concentration was tested (Fig. 8), and the result showed that I reached its maximum value and remained stable when the NaCl volume was > 40 μL. Therefore, a concentration of 40 mmol/L NaCl was chosen.
Figure 4. Scanning electron microscopy. (a) 39.2 nmol/L Apt–GN + Tris/HCl (pH 8.05) + 40 mmol/L NaCl; (b) a + 1.7 nmol/L LYS.
Luminescence 2014; 29: 1003–1007
Figure 8. Effect of NaCl concentration on I368nm 39.2 nmol/L Apt–GN + 3.5 nmol/L LYS + 2.5 mmol/L Tris/HCl (pH 8.05).
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
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Figure 5. Transmission electron microscopy of the Apt–GN.
L. Ma et al. Table 1. The effect of foreign substances Foreign substance
Tolerance (CFS/CLYS)
Glucose L-glutamic L-phenylalanine l-cystine BSA HSA
Relative error (%)
80 80 100 60 40 10
Foreign substance
Tolerance (CFS/CLYS)
Relative (%)
120 60 40 80 60 80
-3.9 -4.9 -4.2 4.3 4.9 3.0
2+
-4.4 -3.1 -4.9 -5.0 -4.0 4.6
Ca K+ Zn2+ Fe3+ Cu2+ Mg2+ Influence of foreign substances
3000
The influence of foreign substances (FS) on the determination of 3.5 nmol/L LYS was examined. The tolerance limit was defined as the molar ratio of [FS]/[LYS] that gives a relative error not more than ± 10%. Table 1 shows that a molar ratio of 100 for L-phenylalanine, 80 for Fe3+, Mg2+, glucose and L-glutamate, 60 for L-cysteine and Cu2+, 40 for bovine serum albumin (BSA), 20 for Zn2+ and Ca2+, and 10 for human serum albumin (HAS) did not interfere with the determination. The method was shown to have good selectivity.
2500 2000 1500 1000 500
Working curve 0 0
1
2
3
4
5
c (n m o l/L ) Figure 9. Working curve.
6
Under the optimal conditions, the working curve was obtained as a plot of the LYS concentration c(nmol/L) vs. ΔI (Fig. 9). The enhanced RRS intensity ΔI at 368 nm was linear for the LYS concentration in the range 0.2–5.2 nmol/L, with a regression
Table 2. Comparison of of the reported assays for LYS Assay
Principle
Linear range DL (nmol/L) (nmol/L)
Colorimetry
LYS combined with GN–Apt probe, GNs were aggregated into a large cluster and the color changed from red to blue. Electrochemistry The combination of LYS and Apt led to the dissociation of double-stranded DNA, and the oxidation behavior of triallylamine changed. SPR GNs were aggregated by LYS that caused the signal to increase. Chemiluminescence Based on competition between LYS and the (Rubpy)2+ 3 cation for the binding sites of Apt in the gold electrode. Fluorescence LYS reacted with single strand DNA binding (SSB)–Apt and releases SSB, binding of the free SSB to a molecule resulted in a turn-on fluorescence signal. RRS GN combined with LYS to form a GN–LYS complex that caused the RRS intensity to increase. RRS LYS reacted with the GN–Apt to form an Apt–LYS complex and release GNs that were aggregated into a large cluster by NaCl.
Comment
Ref.
3.4
Simple, not sensitive
5
–
Sensitive, but complex
6
10–40
7.9
8
0.64–640
0.12
Simple, not sensitive Selective
10
0–15
0.2
Not sensitive
11
5.6–140
2.1
0.2–5.2
0.05
Simple 13 and rapid Simple, rapid This study and sensitive
10–100
0.001–1.1
Table 3. The results for the detection of LYS in samples
1006
Sample
Found (× 10-4 mg/mL)
1 2
8.5, 9.4, 9.8, 10.1, 10.5 11.5, 11.6, 12.4, 12.5, 14.0
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Mean (× 10-4 mg/mL)
LYS in egg (mg/mL)
RSD (%)
9.70 12.4
4.6 6.0
7.9 8.1
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1003–1007
Apt-AuNP RRS method for LYS equation of ΔI = 446c + 49.8, R2 = 0.996, and a detection limit (DL) of 0.05 nmol/L. Compared with previously reported assays (Table 2), this method was simple and sensitive.
Analysis of samples An egg purchased from the market was broken and the egg white was collected. A 500 μL sample of egg white was diluted with 49.5 mL water, and dispersed by ultrasound for 30 min; the solution was collected through a filter paper and was diluted 48 times. The LYS content was analyzed using the procedure described above. The relative standard deviations (RSD) were 7.9 and 8.1% (Table 3).
Conclusions In pH 8.05 Tris/HCl buffer solution, GN was modified by a lysozyme aptamer to obtain a stable Apt–GN probe for LYS. When LYS was added, it combined with the Apt specifically, and released GNs that aggregated to form a large cluster with a RRS peak at 368 nm. The enhanced RRS intensity ΔI was linear to LYS concentration over a certain range. According to these results, a new RRS method has been established for the detection of trace LYS, with good sensitivity, simplicity and rapidity. Acknowledgements This work supported by the National Natural Science Foundation of China (No. 21367005, 21267004, 21165005), the Natural Science Foundation of Guangxi Province of China (No. 2013GXNSFFA019003), and the Research Funds of Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology.
References 1. Alexandria F. On a remarkable bacteriolytic element found in tissues and secretions. Proc R Soc B 1922;93:306–17. 2. Cunningham FE, Proctor VA, Goetsch SJ. Egg-white lysozyme as a food preservative: an overview. World Poultry Sci J 1991;47:141–63. 3. Ishii H, Iwata A, Oka H, Sakamoto N, Ishimatsu Y, Kadota J. Elevated serum levels of lysozyme in desquamative interstitial pneumonia. Int Med 2010;49:874–80. 4. Schroeder S, Stuerenburg HJ, Escherich F, Pfeiffer G, Schroeder F. Lysozyme in ventriculitis: a marker for diagnosis and disease progression. J Neurol Neurosurg Ps 2000;69:696–7. 5. Zhang HG, Wang MJ, Qi HL, Gao Q, Cheng CX. Colorimetric aptamerbased method for the determination of lysozyme using Au nanoparticles. J Northwest Univ 2011;41:617–22. 6. Liu DY, Zhao Y, He XW, Yin XB. Electrochemical aptasensor using the tripropylamine oxidation to probe intramolecular displacement between target and complementary nucleotide for protein array. Biosens Bioelectron 2011;26:2905–10. 7. Chen ZB, Li LD, Zhao HT, Guo L, Mu XJ. Electrochemical impedance spectroscopy detection of lysozyme based on electrodeposited gold nanoparticles. Talanta 2011;85:1501–6. 8. Wang XY, Xu Y, Xu X, Hu K, Xiang MH, Liu LM, et al. Direct determination of urinary lysozyme using surface plasmon resonance lightscattering of gold nanoparticles. Talanta 2010;82:693–7.
9. Wang XY, Xu Y, Cheng Y, Li LM, Liu F, Li N. The gold-nanoparticlebased surface plasmon resonance light scattering and visual DNA aptasensor for lysozyme. Anal Bioanal Chem 2011;400:2085–91. 10. Li Y, Qi HL, Gao Q, Zhang CX. Label-free and sensitive electrogenerated chemiluminescence aptasensor for the determination of lysozyme. Biosens Bioelectron 2011;26:2733–6. 11. Tang D, Liao DL, Zhu QK, Wang FY, Jiao HP, Zhang YJ, et al. Fluorescence turn-on detection of a protein through the displaced singlestranded DNA binding protein binding to a molecular beacon. Chem Commun 2011;47:5485–7. 12. Jiang ZL, Huang GX. Resonance scattering spectra of Micrococcus lysodeikticus and its application to assay of lysozyme activity. Clin Chim Acta 2007;376:136–41. 13. Cai ZX, Yu HF, Ma MH. Determination of lysozyme at the nanogram level in food sample using resonance Rayleigh-scattering method with Au nanoparticles as probe. Spectrochim Acta A 2011;78:1266–71. 14. Wu C, Yang SY, Wu CY, Shen GL, Yu YQ. Split aptamer-based liquid crystal biosensor for ATP assay. Acta Chim Sin 2013;71:367–70. 15. Lin YW, Liu CW, Chang HT. Fluorescence detection of mercury(II) and lead(II) ions using aptamer/reporter conjugates. Talanta 2011;84:324–9. 16. Cheng W, Ding SJ, Li Q, Yu TX, Yin YB, Ju HX, et al. A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension. Biosens Bioelectron 2012;36:12–17. 17. Das BK, Tlili C, Badhulika S, Cella LN, Mulchandani WCA. Singlewalled carbon nanotubes chemiresistor aptasensors for small molecules: picomolar level detection of adenosine triphosphate. Chem Commun 2011;47:3793–5. 18. Sheng WW, Li NB, Luo HQ. Gemini surfactant applied to the heparin assay at the nanogram level by resonance Rayleigh scattering method. Anal Biochem 2012;422:1–6. 19. Tang JX, Peng JD, Zhang L, Xiao Y. High performance liquid chromatography (HPLC) method coupled with resonance Rayleigh scattering detection for the determination of isepamicin. Anal Methods 2012;4:1833–7. 20. Liang AH, Zhou LP, Qin HM, Zhang Y, Ouyang HX, Jiang ZL. A highly sensitive aptamer-nanogold catalytic resonance scattering spectral assay for melamine. J Fluoresc 2011;21:1907–12. 21. Jiang ZL, Xu LL, Wang LS, Liang AH, Jiang ZL. A simple and sensitive label-free immunoassay for factor B using resonance scattering spectral detection. Chin J Chem 2012;30:1636–40. 22. Jiang ZL, Yao DM, Li F, Liang AH. Resonance scattering assay for trace human chorionic gonadotrophin using gold–platinum nanoalloy immunoprobe as catalyst. Acta Chim Sin 2012;70:1748–54. 23. Huang ST, Wang GL, Li NB, Luo HQ. Mechanism of the pH-induced aggregation reaction between melamine and phosphate. RSC Adv 2012;2:10948–51. 24. Liang AH, Wei YY, Wen GQ, Yin WQ, Jiang ZL. A new resonance Rayleigh scattering method for trace Pb, coupling the hydride generation reaction with nanogold formation. RSC Adv 2013;3:12585–8. 25. Jiang ZL, Fan YY, Chen ML, Liang AH, Liao XJ, Wen GQ, et al. Resonance scattering spectral detection of trace Hg2+ using aptamermodified nanogold as probe and nanocatalyst. Anal Chem 2009;81:5439–45. 26. Liang AH, Ouyang HX, Jiang ZL. Resonance scattering spectral detection of trace ATP based on label-free aptamer reaction and nanogold catalysis. Analyst 2011;136:4514–19. 27. Liang AH, Liu QY, Wen GQ, Jiang ZL. The surface-plasmon-resonance effect of nanogold/silver and its analytical applications. Trans Anal Chem 2012;37:32–47. 28. Huang SW, Li JS, Liang AH, Jiang ZL. Highly sensitive and selective detection of thrombin by Aptamer-modified nanosilver resonance scattering spectral probe. Acta Chim Sin 2011;69:183–9.
1007
Luminescence 2014; 29: 1003–1007
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
Revised: 3 November 2013,
Accepted: 18 January 2014
Published online in Wiley Online Library: 10 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2650
A new and highly sensitive resonance Rayleigh scattering assay for lysozyme using aptamer– nanogold as a probe Lu Ma, Xinghui Zhang, Aihui Liang, Qingye Liu and Zhiliang Jiang* ABSTRACT: Gold nanoparticles (GN), 10 nm in size, were modified by using lysozyme aptamer (Apt) to obtain a stable Apt–GN probe in pH 8.05 Tris/HCl buffer solutions containing 0.04 mol/L NaCl. Upon addition of lysozyme (LYS), it reacted with the Apt of the probe to form a very stable Apt–LYS complex and to release GNs, which aggregated to form large clusters with a resonance Rayleigh scattering (RRS) peak at 368 nm. The enhanced peak intensity, ΔI, was linear to the LYS concentration in the range 0.2–5.2 nmol/L, with a detection limit of 0.05 nmol/L. The influence of foreign substance was tested, and the results showed that this RRS method has high selectivity. This Apt–GN RRS method was applied to the analysis of LYS in a real sample, with satisfactory results. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: lysozyme; aptamer; gold nanoparticle; resonance Rayleigh scattering
Introduction
Luminescence 2014; 29: 1003–1007
Experimental Apparatus and reagents A F-7000 Hitachi fluorescence spectrometer (Hitachi Co., Tokyo, Japan), a TU-1901 double-beam UV/vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China), a JSM-6380 LV scanning electron microscope (Japan), a JEM-2100 F field emission transmission electron microscope (Japan), a 79-1 magnetic heating stirrer (Zhongda Instrumental Plant, Jiangsu, China), and a SK8200LH ultrasonic reactor (Kedao Ultrasonic Instruments Ltd, Shanghai, China) were used. A 3.49 μmol/L stock solution of LYS was prepared by dissolving 0.0500 g of LYS in 10 mL of water, and a 0.174 μmol/L LYS working solution was used. LYS aptamer (Sangon Biotech Co., Ltd.) with a sequence of 5′-CAGTGTATCGAATTCATCAGGGCTA AAGAGTGCAGAGTTACT-3′ was used at a concentration of 100 μmol/L; 2.0 mol/L NaCl, 1.0% HAuCl4 and 1.0% trisodium citrate were also used. The gold nanoparticle (GN) was synthesized by reduction of HAuCl4 using trisodium citrate. Doubly distilled water (50 mL) was added to a flask and brought to the boil under stirring. * Correspondence to: Z. Jiang, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Conservation of Education Ministry and Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, Guangxi Normal University, Guilin 541004, China. Tel: +86-07735846141; Fax: +86-07735846201. E-mail: [email protected] Key Laboratory of Ecology of Rare and Endangered Species and Environmental Conservation of Education Ministry and Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, Guangxi Normal University, Guilin541004, China
Copyright © 2014 John Wiley & Sons, Ltd.
1003
Lysozyme (LYS), also known as muramidase or N-acetylmuramide glycanohydrolase, is an alkaline globulin that consists of 129 amino acid residues, and is a powerful bactericide found in nasal mucus by Fleming in 1922 (1). It exists in mammalian body fluids including tears, saliva, plasma, urine, milk, microorganisms and egg white at a high level. LYS has antimicrobial and bactericidal effects; it selectively dissolves the outer membrane of bacteria, destroying their bioactivity. Because it is safe and non-toxic, LYS has been used as a biological preservative in the food industry (2). In addition, LYS also has anti-inflammatory, anti-virus, immune-boosting and other pharmacological activities, and it has also been applied as a potential marker in the diagnosis of disease (3,4). Therefore, it is important to develop a simple, rapid and sensitive method for the determination of LYS. To date, several methods have been used to determine LYS, including spectrophotometry (5), electrochemistry (6,7), surface plasmon resonance (SPR) (8,9), chemiluminescence (10), fluorescence (11) and resonance Rayleigh scattering (RRS) (12,13). However, these methods have some limitations due to their complicated modes of operation, poor sensitivity or poor selectivity. As aptamer (Apt) is selected from a synthetic and random sequence library in vitro via a combinatorial chemistry technique known as SELEX. Advantages of SELEX include ease of preparation, high stability, ease of modification, and a wide range of binding target molecules such as metal ions, small molecules, proteins and bacteria (14–17). It has been an important development in analytical chemistry. RRS spectroscopy is sensitive, rapid and simple, and has been used in a wide range of applications in different fields such as biochemistry, analytical chemistry and nanomaterial research (18–24). Combined with Apt-modified nanogold (GN), RRS has been used in the detection of metal ions (25), melamine, ATP (26,27) and thrombin (28). As far as we know, there is no report of the use of RRS for the detection of LYS, based on an Apt-modified GN probe. In this paper, a novel and simple
Apt–GN RRS assay was proposed to detect LYS, based on the Apt–GN reaction and the RRS effect of GN aggregates.
L. Ma et al. 3500 3000
Intensity (a.u.)
Then, 3.5 mL of 1% trisodium citrate and 0.5 mL of 1% HAuCl4 were added rapidly to the boiling water. After boiling for 10 min with stirring, the color changed form lilac to purple to red. The mixture was cooled to room temperature with stirring, and then diluted to 50 mL. The GN concentration was 58.0 μg/mL. Apt–GN was prepared as follows: 2.0 mL of a 58.0 μg/mL GN solution was added to 0.8 mL of a 1 μmol/L Apt solution, mixed well and set aside for 10 min. The Apt–GN concentration, calculated as Apt, was 0.28 μmol/L. All reagents were of analytical grade and the water was doubly distilled.
g
2500 2000
a
1500 1000 500 0 200
Procedure
300
400
500
600
Wavelength (nm)
Results and discussion Principle Apt combined with GN to form the Apt–GN probe via electrostatic forces, van der Waal’s forces and intermolecular forces, and the probe was stable in high-concentration salts. Upon addition, LYS reacted with the probe to form a very stable Apt–LYS complex and to release GNs. Under the action of NaCl, the GNs aggregated to form large clusters that caused the enhancement of RRS intensity. The number of released GNs increased as the concentration of LYS increased, the GN aggregates also became larger, and the RRS intensity increased accordingly. Based on this, a RRS assay could be developed for the detection of trace LYS (Fig. 1). RRS spectra In pH 8.05 Tris/HCl buffer solution and in the presence of NaCl, the RRS signal of the Apt–GN probe was weak (Fig. 2). Upon addition of LYS, the probe combined with LYS specifically, and the released NGs aggregated to form a large cluster, the color changed from red to purple, and the RRS intensity increased at 285, 368 and 525 nm (Fig. 2). Because the light sources in the apparatus have strong emission at wavelengths of 285 and 368 nm, the system has two RRS peaks caused by the light source at both wavelengths, and the peak at 525 nm was due
Figure 2. RRS spectra of LYS–Apt–GN systems. (a) 39.2 nmol/L Apt–GN + Tris/HCl (pH 8.05) + 40 mmol/L NaCl; (b) a + 0.4 nmol/L LYS; (c) a + 1.7 nmol/L LYS; (d) a + 2.6 nmol/L LYS; (e) a + 3.5 nmol/L LYS; (f) a + 4.4 nmol/L LYS; (g) a + 5.2 nmol/L LYS.
to the GNs. The peak at 525 nm is not sensitive, whereas the peak at 368 nm is most sensitive and has good linearity. Thus, a wavelength of 368 nm was chosen. Absorption spectra There is only an SPR absorption peak at 519 nm for GN particles. After modification by Apt, the SPR peak does not change (Fig. 3a), and the absorption is very low due to the low concentration of NGs (5.8 μg/mL Au). Upon addition, LYS reacted with the Apt in the probe to form a stable complex, which exhibited a new absorption peak at 620 nm. As the concentration of LYS was increased, the absorption increased at about 620 nm (Fig. 3). The red-shift and broadening of the SPR peak showed that NGs were aggregated into large clusters in the solution system. Electron microscope A 1.5 mL aptamer reaction solution was obtained, and centrifuged for 20 min at 15,000 rpm. After the supernatant had been removed, the precipitate was diluted with 1.5 mL water and dispersed by ultrasound for 30 min; the process was repeated twice. A 2.0 μL aliquot of solution was added to a silicon chip and observed by scanning electron microscopy. It 0.14 0.12 0.1
Abs
Two hundred and eighty microliters of 0.28 μmol/L Apt–GN, 50 μL of Tris/HCl (pH 8.05) and a known volume of LYS solution were added to a 5 mL marked test tube and the mixture was diluted to 1.0 mL, then 40 μL of 2.0 mol/L NaCl was added and the mixture was diluted to 2.0 mL. RRS spectra were recorded by synchronous scanning of the excited wavelength λex and emission wavelength λem (λex – λem = Δλ = 0), a photomultiplier tube (PMT) voltage of 450 V was used, and excited and emission slit width of 5 nm were used on the fluorescence spectrophotometer. The RRS intensity at 368 nm (I368) and the blank value (I368)0 without LYS were recorded. The value of ΔI = I368 – (I368)0 was calculated.
0.08 0.06 0.04 0.02 0 300
400
500
600
700
800
wavelength (nm)
1004
Figure 1. Principle of the Apt–RRS method for LYS.
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Figure 3. Absorption spectra of LYS–Apt–GN systems. (a) 39.2 nmol/L Apt–GN + Tris/HCl (pH 8.05) + 40 mmol/L NaCl; (b) a + 0.4 nmol/L LYS; (c) a + 1.7 nmol/L LYS; (d) a + 3.5 nmol/L LYS; (e) a + 4.4 nmol/L LYS; (f) a + 5.2 nmol/L LYS.
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1003–1007
Apt-AuNP RRS method for LYS 3500 3000 2500 2000
I
was shown that the Apt–GN probe was dispersed in solution (Fig. 4a), LYS reacted with the Apt in the probe to form a stable complex and to release GNs, which aggregated to form large clusters under the action of NaCl (Fig. 4b). Transmission electron microscopy (Fig. 5) also indicated that the Apt–GN probe is spherical, with an average size of 10 nm and is dispersed in solution.
1500
Preparation of Apt–GN and its concentration selection A 200 μL aliquot of a 58.0 μg/mL GN solution was used to test the impact of Apt on the system I368. The Apt was not enough to protect GN when the Apt concentration was < 40 nmol/L, and GNs were aggregated into a large cluster that caused the increase in RRS intensity (Fig. 6). When the Apt concentration was 40 nmol/L, it was just enough to modify GN, and the Apt–GN complex was stable in solution. Thus, 40 nmol/L was the minimum amount of Apt that stabilized 200 μL GN. The effect of the Apt–GN concentration was also considered, and 280 μL of 0.28 μmol/L Apt–GN was chosen for use.
1000 500 0
10
20
30
40
50
c (nmol/L) Figure 6. Effect of the Apt concentration on ΔI368nm 5.8 μg/mL GN + 2.5 mmol/L Tris/HCl (pH 8.05) + 40 mmol/L NaCl.
Effect of pH Buffer solutions including acetate, phosphate and Tris/HCl were examined. Tris/HCl buffer solution, giving biggest ΔI value, was selected for use. The effect of pH on the system was tested (Fig. 7). When the pH was 8.05, the ΔI of the system was maximal, so pH 8.05 was chosen for use. The effect of Tris/HCl concentration was also tested, when the Tris/HCl concentration was 1.25 mmol/L, ΔI reached its maximum value. Therefore, a 1.25 mmol/L pH 8.05 Tris/HCl buffer was chosen.
a
Figure 7. Effect of pH on ΔI368nm 39.2 nmol/L Apt–GN + 3.5 nmol/L LYS + 40 mmol/L NaCl.
b
Effect of NaCl concentration The effect of NaCl concentration was tested (Fig. 8), and the result showed that I reached its maximum value and remained stable when the NaCl volume was > 40 μL. Therefore, a concentration of 40 mmol/L NaCl was chosen.
Figure 4. Scanning electron microscopy. (a) 39.2 nmol/L Apt–GN + Tris/HCl (pH 8.05) + 40 mmol/L NaCl; (b) a + 1.7 nmol/L LYS.
Luminescence 2014; 29: 1003–1007
Figure 8. Effect of NaCl concentration on I368nm 39.2 nmol/L Apt–GN + 3.5 nmol/L LYS + 2.5 mmol/L Tris/HCl (pH 8.05).
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1005
Figure 5. Transmission electron microscopy of the Apt–GN.
L. Ma et al. Table 1. The effect of foreign substances Foreign substance
Tolerance (CFS/CLYS)
Glucose L-glutamic L-phenylalanine l-cystine BSA HSA
Relative error (%)
80 80 100 60 40 10
Foreign substance
Tolerance (CFS/CLYS)
Relative (%)
120 60 40 80 60 80
-3.9 -4.9 -4.2 4.3 4.9 3.0
2+
-4.4 -3.1 -4.9 -5.0 -4.0 4.6
Ca K+ Zn2+ Fe3+ Cu2+ Mg2+ Influence of foreign substances
3000
The influence of foreign substances (FS) on the determination of 3.5 nmol/L LYS was examined. The tolerance limit was defined as the molar ratio of [FS]/[LYS] that gives a relative error not more than ± 10%. Table 1 shows that a molar ratio of 100 for L-phenylalanine, 80 for Fe3+, Mg2+, glucose and L-glutamate, 60 for L-cysteine and Cu2+, 40 for bovine serum albumin (BSA), 20 for Zn2+ and Ca2+, and 10 for human serum albumin (HAS) did not interfere with the determination. The method was shown to have good selectivity.
2500 2000 1500 1000 500
Working curve 0 0
1
2
3
4
5
c (n m o l/L ) Figure 9. Working curve.
6
Under the optimal conditions, the working curve was obtained as a plot of the LYS concentration c(nmol/L) vs. ΔI (Fig. 9). The enhanced RRS intensity ΔI at 368 nm was linear for the LYS concentration in the range 0.2–5.2 nmol/L, with a regression
Table 2. Comparison of of the reported assays for LYS Assay
Principle
Linear range DL (nmol/L) (nmol/L)
Colorimetry
LYS combined with GN–Apt probe, GNs were aggregated into a large cluster and the color changed from red to blue. Electrochemistry The combination of LYS and Apt led to the dissociation of double-stranded DNA, and the oxidation behavior of triallylamine changed. SPR GNs were aggregated by LYS that caused the signal to increase. Chemiluminescence Based on competition between LYS and the (Rubpy)2+ 3 cation for the binding sites of Apt in the gold electrode. Fluorescence LYS reacted with single strand DNA binding (SSB)–Apt and releases SSB, binding of the free SSB to a molecule resulted in a turn-on fluorescence signal. RRS GN combined with LYS to form a GN–LYS complex that caused the RRS intensity to increase. RRS LYS reacted with the GN–Apt to form an Apt–LYS complex and release GNs that were aggregated into a large cluster by NaCl.
Comment
Ref.
3.4
Simple, not sensitive
5
–
Sensitive, but complex
6
10–40
7.9
8
0.64–640
0.12
Simple, not sensitive Selective
10
0–15
0.2
Not sensitive
11
5.6–140
2.1
0.2–5.2
0.05
Simple 13 and rapid Simple, rapid This study and sensitive
10–100
0.001–1.1
Table 3. The results for the detection of LYS in samples
1006
Sample
Found (× 10-4 mg/mL)
1 2
8.5, 9.4, 9.8, 10.1, 10.5 11.5, 11.6, 12.4, 12.5, 14.0
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Mean (× 10-4 mg/mL)
LYS in egg (mg/mL)
RSD (%)
9.70 12.4
4.6 6.0
7.9 8.1
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Luminescence 2014; 29: 1003–1007
Apt-AuNP RRS method for LYS equation of ΔI = 446c + 49.8, R2 = 0.996, and a detection limit (DL) of 0.05 nmol/L. Compared with previously reported assays (Table 2), this method was simple and sensitive.
Analysis of samples An egg purchased from the market was broken and the egg white was collected. A 500 μL sample of egg white was diluted with 49.5 mL water, and dispersed by ultrasound for 30 min; the solution was collected through a filter paper and was diluted 48 times. The LYS content was analyzed using the procedure described above. The relative standard deviations (RSD) were 7.9 and 8.1% (Table 3).
Conclusions In pH 8.05 Tris/HCl buffer solution, GN was modified by a lysozyme aptamer to obtain a stable Apt–GN probe for LYS. When LYS was added, it combined with the Apt specifically, and released GNs that aggregated to form a large cluster with a RRS peak at 368 nm. The enhanced RRS intensity ΔI was linear to LYS concentration over a certain range. According to these results, a new RRS method has been established for the detection of trace LYS, with good sensitivity, simplicity and rapidity. Acknowledgements This work supported by the National Natural Science Foundation of China (No. 21367005, 21267004, 21165005), the Natural Science Foundation of Guangxi Province of China (No. 2013GXNSFFA019003), and the Research Funds of Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology.
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